Gene therapy for haploinsufficiency

Abstract

Methods and compositions are provided for activating transcription in a mammalian cell.

What is claimed is: 
     
       1. A method of treating a haploinsufficiency disease in a mammalian subject, the method comprising contacting a cell of the subject with a composition comprising:
 i) a catalytically inactive CRISPR nuclease fused to a transcriptional activation domain, and 
 ii) a guide RNA, wherein the guide RNA comprises:
 a) a targeting region that, under conditions present in a nucleus of the cell, specifically hybridizes to a promoter region or an enhancer region operably linked to a wild-type copy of a haploinsufficient gene; and 
 b) a binding region that specifically binds the catalytically inactive CRISPR nuclease under conditions present in a nucleus of the cell, 
 
 wherein the contacting forms a complex comprising the catalytically inactive CRISPR nuclease bound to the guide RNA, wherein the targeting region of the guide RNA in the complex is hybridized to the promoter or enhancer of the wild-type copy of the haploinsufficient gene; and 
 wherein the complex activates transcription of the wild-type copy of the haploinsufficient gene in an amount and for a duration sufficient to treat the haploinsufficiency disease in the subject. 
 
     
     
       2. The method of  claim 1 , wherein the contacting comprises:
 (a) contacting the cell with an episomal vector encoding the guide RNA or the catalytically inactive CRISPR nuclease; or 
 (b) contacting the cell with an episomal vector encoding the guide RNA and the catalytically inactive CRISPR nuclease; or 
 (c) contacting the cell with an episomal vector encoding the guide RNA and a second episomal vector encoding the catalytically inactive CRISPR nuclease; or 
 (d) injection of nucleic acid encoding the guide RNA and/or the catalytically inactive CRISPR nuclease into a region of a brain containing a hypothalamus; or 
 (e) injection of an adeno-associated viral vector comprising nucleic acid encoding the guide RNA and/or the catalytically inactive CRISPR nuclease into a region of a brain containing a hypothalamus. 
 
     
     
       3. The method of  claim 2 , wherein the episomal vector(s):
 (a) are non-integrating; and/or 
 (b) are non-replicating; and/or 
 (c) are adeno-associated virus (AAV) vectors; and/or 
 (d) independently comprise a first and a second end, wherein the first end and second end each independently comprise an AAV inverted terminal repeat. 
 
     
     
       4. The method of  claim 1 , wherein the catalytically inactive CRISPR nuclease comprises (i) a nuclease domain that has been modified to eliminate nuclease and nicking activity and (ii) a transcriptional activation domain, and/or a D10A, H840A  S. pyogenes  dCas9. 
     
     
       5. The method of  claim 1 , wherein the catalytically inactive CRISPR nuclease is a catalytically inactive CRISPR nuclease-VP64 fusion polypeptide. 
     
     
       6. The method of  claim 1 , wherein the haploinsufficient gene is SCN1A, SCN2A, SIM1, or MC4R. 
     
     
       7. The method of  claim 1 , wherein the cell is a non-dividing cell, a neuron, or a hypothalamus cell. 
     
     
       8. The method of  claim 1 , wherein the haploinsufficiency disease is selected from the group consisting of obesity, autism, epilepsy, intellectual disability, aniridia, and polycystic kidney disease.